Crystal Structure, Mutations, and Catalytic Properties of 3-Hexulose-6-phosphate Synthase from <i>Pyrococcus horikoshii</i>

3-Hexulose-6-phosphate synthase, a key enzyme in the ribulose monophosphate pathway, plays a central role in formaldehyde assimilation and detoxification, offering great potential for third-generation green biomanufacturing. In this study, we determined the crystal structure of HPS from <i>Pyrococcus horikoshii</i> at a 2.64 Å resolution. Combined approaches, including molecular docking, multiple sequence alignment, and alanine scanning, identified critical catalytic residues. Two variants, T136C and V186W, exhibited over 6-fold higher activity than the wild type. Molecular dynamics simulations indicated increased structural rigidity and enhanced stability upon substrate (d-ribulose-5-phosphate) binding, along with significantly improved binding energies. Furthermore, an enzyme cascade converting d-xylose and formaldehyde to d-fructose-6-phosphate was constructed to evaluate the HPS activity under optimized conditions. These findings provide insights into the catalytic mechanism of HPS and offer a basis for its application in one-carbon bioconversion.